bioRxiv preprint doi: https://doi.org/10.1101/2020.09.01.278846; this version posted September 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 SPECIES-DEPENDENT HETEROPHILIC AND HOMOPHILIC CADHERIN INTERACTIONS IN 2 INTESTINAL INTERMICROVILLAR LINKS 3 4 Michelle E. Gray*1, Zachary R. Johnson*§2, Debadrita Modak*2, Matthew J. Tyska3, and Marcos Sotomayor#1,2 5 1Ohio State Biochemistry Program 6 2Department of Chemistry and Biochemistry 7 The Ohio State University, 484 W 12th Avenue, Columbus, OH 43210 8 3Department of Cell and Developmental Biology 9 Vanderbilt University School of Medicine, Nashville, TN 37232 10 *Co-first authors 11 §Current address: Marshall University, Joan C. Edwards School of Medicine, Huntington, WV 25701 12 # Corresponding Author 13 E-mail: [email protected] 14 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.01.278846; this version posted September 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 ABSTRACT 2 Enterocytes are specialized epithelial cells lining the luminal surface of the small intestine that build densely packed 3 arrays of microvilli known as brush borders. These microvilli drive nutrient absorption and are arranged in a hexagonal 4 pattern maintained by intermicrovillar links formed by two non-classical members of the cadherin superfamily of calcium- 5 dependent cell adhesion proteins: protocadherin-24 (PCDH24, also known as CDHR2) and the mucin-like protocadherin 6 (CDHR5). The extracellular domains of these proteins are involved in heterophilic and homophilic interactions important 7 for intermicrovillar function, yet the structural determinants of these interactions remain unresolved. Here we present X- 8 ray crystal structures of the PCDH24 and CDHR5 extracellular tips and analyze their species-specific features relevant for 9 adhesive interactions. In parallel, we use binding assays to identify the PCDH24 and CDHR5 domains involved in both 10 heterophilic and homophilic adhesion for human and mouse proteins. Our results suggest that homophilic and 11 heterophilic interactions involving PCDH24 and CDHR5 are species dependent with unique and distinct minimal 12 adhesive units. 13 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.01.278846; this version posted September 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 INTRODUCTION 2 Enterocytes are specialized epithelial cells lining the luminal surface of the small intestine and are fundamental players in 3 nutrient absorption with a role in host defense (Maroux et al., 1988; Mukherjee et al., 2008; Selsted and Ouellette, 2005; 4 Shifrin and Tyska, 2012). Key to the aforementioned processes is the brush border, so named because the apical surface 5 of the enterocytes is coated by thousands of microvilli of similar length and size organized in a hexagonal arrangement 6 (Mooseker, 1985). The organization and structure of the microvilli is maintained by a network of cytoplasmic and 7 transmembrane proteins known as the intermicrovillar adhesion complex (IMAC), which includes proteins with 8 extracellular adhesive domains and cytoplasmic parts tethered to the actin cytoskeleton that forms the interior of the 9 microvilli (Crawley et al., 2016, 2014b, 2014a; Li et al., 2016; Pinette et al., 2018; Weck et al., 2016). Perturbations of 10 brush border function are often associated with disease (Bailey et al., 1989; Khubchandani et al., 2011; Vallance et al., 11 2002; Wilson et al., 2001) and, not surprisingly, disruption of the IMAC components results in intestinal dysfunction 12 (Bitner-Glindzicz et al., 2000; Crawley et al., 2014b; Hussain et al., 2004). 13 14 The extracellular parts of the IMAC stem from a pair of cellular adhesion proteins that belong to the cadherin superfamily, 15 protocadherin-24 (PCDH24, also known as CDHR2) and mucin-like protocadherin (CDHR5) (Crawley et al., 2014b; 16 McConnell et al., 2011). These two non-classical protocadherins form intermicrovillar links essential for brush border 17 morphogenesis and function (Crawley et al., 2014a, 2014b), stabilizing the microvillar hexagonal patterns as shown by 18 freeze-etch electron microscopy and PCDH24 immuno-labeling combined with transmission electron microscopy 19 (Crawley et al., 2014b). Mutations that impair the PCDH24 and CDHR5 interaction, along with knockdowns of PCDH24 20 and CDHR5, cause a remarkable reduction in microvillar clustering ex vivo (Crawley et al., 2014b). A PCDH24 knockout 21 mouse model was found to be viable but body weight of mutant mice was lower than wild-type and there were defects in 22 the packing of the microvilli in the brush border (Pinette et al., 2018). Despite the important physiological role played by 23 PCDH24 and CDHR5, little is known about the molecular details of how these proteins interact to form the 24 intermicrovillar links. 25 26 PCDH24 and CDHR5 are members of a large superfamily of proteins with extracellular domains that often mediate 27 adhesion and that have contiguous and similar, but not identical, extracellular cadherin (EC) repeats. The linker regions 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.01.278846; this version posted September 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 between the EC repeats typically bind three calcium ions essential for adhesive function (Frank and Kemler, 2002; 2 Gumbiner, 2005; Sano et al., 1993; Takeichi, 1990). PCDH24 belongs to the Cr-2 subfamily and has 9 EC repeats, a 3 membrane adjacent domain (MAD10), a transmembrane domain, and a C-terminal cytoplasmic domain (Bork and Patthy, 4 1995; De-la-Torre et al., 2018; Ge et al., 2018; Hulpiau and van Roy, 2009; McConnell et al., 2011; Pei and Grishin, 5 2017). CDHR5 belongs to the Cr-3 subfamily and has 4 EC repeats, a mucin-like domain (MLD), a transmembrane 6 domain, and a C-terminal cytoplasmic domain. Some human CDHR5 isoforms lack the MLD, which is not essential for 7 the interaction with human PCDH24 (Crawley et al., 2014b). Sequence alignments of the N-terminal repeats (EC1-3) 8 suggest that PCDH24 and CDHR5 are similar to another pair of heterophilic interacting cadherins: cadherin-23 (CDH23) 9 and protocadherin-15 (PCDH15), the cadherins responsible for the formation of inner-ear tip links (Elledge et al., 2010; 10 Kazmierczak et al., 2007; Sotomayor et al., 2014, 2012, 2010). PCDH24 is most similar to CDH23, having the residues 11 and elongated N-terminal β-strand that are predicted to favor the formation of an atypical calcium-binding site at its tip 12 (Elledge et al., 2010; Sotomayor et al., 2010). CDHR5 is most similar to PCDH15, and features cysteine residues 13 predicted to form a disulfide bond at its tip (Sotomayor et al., 2014, 2012). In addition, a mutation in CDHR5, R84G 14 (residue numbering throughout the text corresponds to processed proteins, see Methods), which mimics a deafness-related 15 mutation in PCDH15, interferes with the intermicrovillar links formed by PCDH24 and CDHR5 (Crawley et al., 2014b), 16 suggesting that these proteins interact in a similar fashion to the heterophilic tip-link “handshake” formed by CDH23 and 17 PCDH15 (Behrendt et al., 2012; Sotomayor et al., 2014, 2012). 18 19 To better understand how PCDH24 and CDHR5 interact to form intermicrovillar links, we used a combination of 20 structural and bead aggregation assays to characterize the adhesive properties and mechanisms of these cadherin family 21 members. We present the X-ray crystallographic structures of Homo sapiens (hs) PCDH24 EC1-2, Mus musculus (mm) 22 PCDH24 EC1-3, and hs CDHR5 EC1-2 refined at 2.3 Å, 2.1 Å, and 1.9 Å resolution, respectively. The structures give 23 insight into possible binding mechanisms among species, which are further probed by bead aggregation assays revealing 24 that the human and mouse intermicrovillar cadherins do not engage in the same homophilic and heterophilic interactions. 25 Based on these results, we suggest models for the PCDH24/CDHR5 heterophilic interaction utilized to form 26 intermicrovillar links. 27 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.01.278846; this version posted September 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 RESULTS 2 PCDH24 and CDHR5 tip sequences are poorly conserved across species 3 Intermicrovillar links formed by PCDH24 and CDHR5 in the enterocyte brush border are similar to inner-ear tip links 4 formed by CDH23 and PCDH15. Both types of links are essential for the development, assembly, and function of actin- 5 based structures (microvilli in the gut and stereocilia in the inner ear), and both are made of long cadherin proteins that 6 have similar cytoplasmic partners involved in interactions with the cytoskeleton and in signaling (Crawley et al., 2014a; 7 Pepermans and Petit, 2015). Sequence analyses have revealed similarities between the tips of PCDH24 and CDH23, and 8 between the tips of CDHR5 and PCDH15 (Sotomayor et al., 2014).